Mesoporous platinum electrode and method for detecting biochemical substrate using the mesoporous platinum electrode

ABSTRACT

The present invention relates to a mesoporous platinum electrode for detecting biochemical substrate, comprising an electrode and a mesoporous platinum layer covering the surface thereof, and a method for detecting a biochemical substrate using the mesoporous platinum electrode. Using the present invention, glucose concentration can be selectively determined while excluding interference of interfering agents.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates to a mesoporous platinum electrode and amethod for detecting a biochemical substrate using it, and moreparticularly to a mesoporous platinum electrode including an electrodewith a mesoporous platinum layer covering a surface thereof, and amethod for quantify the concentration of -glucose by detecting aselective response to the glucose oxidation reaction using themesoporous platinum electrode.

(b) Description of the Related Art

A biosensor, in combination with electric devices, converts chemicalinformation in a biological sample into an electrical signal, which canbe easily treated. Biosensors are widely developed and applied in themedical field, due to the advantages of real-time selective monitoringof quantitative information of an analyte without complicated chemicaland biological pre-treatments.

The study of biosensors for glucose has been extensively carried out forthe purpose of glucose concentration monitoring for diabetes. Thebiosensor for glucose detection measures the concentration of glucose(analyte) using the enzyme layer that is immobilized in the confinedregion, generally on an electrode. Enzymatic glucose biosensors usingenzyme have been studied extensively and developed in various types.However, there has been limited application because of enzymeinstability. The temperature and pH affect severely on the activity ofenzyme.

The studies on non-enzymatic biosensors for glucose detection have beencarried out (Vassilyev, Y. B., Khazova, O. A., Nikolaeva, N. N. J.Electroanal. Chem. 1985, 196, 105; Beden, B., Largeaud, F., Kokoh, K.B., Lamy, C. Anal. Chem. 1996, 41, 701; Bae, I. T., Yeager, E., Xing,X., Liu, C. C. J. Electroanal. Chem. 1991, 309, 131; Sakamoto, M.,Takamura, K. Bioelectrochem. Bioener. 1982, 9, 571; Kokkinidis, G.,Xonoglou, N. Bioelectrochem. Bioener. 1985, 14, 375; Wittstock, G.,Strubing, A., Szargan, R., Werner, G. J. Electroanal. Chem. 1998, 444,61-73; Zhang, X., Chan, K.-Y., You, J.-K., Lin, Z.-G., Tseung, A. C. C.J. Electroanal. Chem. 1997, 430, 147-153; Sun, Y., Buck, H., Mallouk, T.E. Anal. Chem. 2001, 73, 1599-1604; Shoji, E., Freund, M. S. 2001, 123,3383-3384). However, most of the non-enzymatic glucose sensors studiedundergo interference by ascorbic acid (AA), uric acid, and4-acetamidophenol (AP), which are important interfering species.

An example of a non-enzymatic glucose biosensor is one that utilizes thedirect oxidation of glucose on a platinum surface (Anal. Chem. 2001, 73,1599-1604). In the direct oxidation on the platinum electrode, theoxidation rate of glucose is much lower than that of interferingspecies, so it is very difficult to construct a non-enzymaticamperometric sensor using platinum on an electrode.

A possible method to alleviate the problems met by platinum is to use aPt-Pb alloy electrode (Pt₂Pb electrodes). Compared with pure platinumsurfaces, glucose is electrochemically oxidized on Pt₂Pb surfaces atremarkably negative potentials, and Pt₂Pb is relatively insensitive tointerfering species such as L-ascorbic acid (AA), uric acid,4-acetamidophenol (AP), and so on. Moreover, Pt₂Pb operates more stablydue to insoluble Pb and larger responses than pure Pt. However, in spiteof these valuable advantages, surface poisoning by chloride ions remainsa serious problem, in which the amperometric signal diminishes rapidlyin the presence of 0.01 N NaCl and eventually almost disappears.

The modification of platinum surfaces with other materials has also beenattempted. Even though platinum surfaces modified by Tl, Pb, Bi, or WO₃reportedly show catalytic activity for glucose oxidation, thedissolution of metal ions and the toxicity of the heavy metal elementsinvolved prevent these methods from being put to practical use.

Mesoporous materials have a pore size between 2 and 50 nm, and a micellstructure or a liquid crystal structure consisting of surfactantsinduces the pore structure of the mesoporous materials. The surfactantsconsist of a hydrophilic head group and a hydrophobic tail group, andvarious self-assembled micell and liquid crystal structures arecomprised of the surfactants in aqueous solution. Organic/inorganicnanocomposites are formed by interaction between the hydrophilic groupof the surface of the micell or liquid crystal structure and theinorganic material, and mesoporous materials are obtained by extractionof surfactants. Mesoporous platinum was fabricated by this principle,and studies on characteristics thereof have been performed (e.g. Gollas,B., Elliott, J. M., Bartlett, P. N. Electrochimica Acta 2000, 45,3711-3724; Attard, G. S., Glyde, J. C., Goeltner, C. G. Nature 1995,378, 366-368; Attard, G. S., Goeltner, C. G., Corker, J. M., Henke, S.,Templer, R. H. Angew. Chem. Int. Ed. 1997, 36, 1315; Whitehead, A. H.,Elliott, J. M., Owen, J. R., Attard, G. S. Chem. Commun. 1999, 331-332;Attard, G. S., Edgar, M., Goeltner, C. G. Acta Mater. 1998, 46, 751-758;Birkin, P. R., Elliott, J. M., Watson, Y. E. Chem. Commun. 2000,1693-1694; Elliott, J. M., Owen, J. R. Phys. Chem. Chem. Phys. 2000, 2,5653-5659). Mesoporous platinum film was initially produced byelectrodeposition from a hexagonal (H₁) liquid crystalline phasecomposed of the non-ionic surfactant (octaethylene glycol monohexadecylether, C₁₆EO₈) (e.g. Attard, G. S., Bartlett, P. N., Coleman, N. R. B.,Elliott, J. M., Owen, J. R., Wang, J. H. Science 1997, 278, 838-840).Reportedly, the electrodeposited platinum film with cylindricalhexagonally arrayed pores (pore diameter, 2.5 nm; pore-pore distance,5.0 nm) was adherent and shiny. According to Evans et al. (Evans, S. A.G., Elliott, J. M., Andrews, L. M., Bartlett, P. N., Doyle, P. J.,Denuault, G. Anal. Chem. 2002, 74, 1322-1326), mesoporous Pt films(Elliott, J. M., Birkin, P. R., Bartlett, P. N., Attard, G. S. Langmuir1999, 15, 7411-7415) electrodeposited onto microelectrodes showedtremendous improvements in hydrogen peroxide detection sensitivitycompared with bare platinum.

SUMMARY OF THE INVENTION

In order to alleviate the drawbacks of previous technology, and it is anobject of the present invention to provide a non-enzymatic andheavy-metal-free detection method for glucose.

It is another object of the present invention to provide a non-enzymaticmethod of selectively detecting glucose oxidation using mesoporousplatinum.

To accomplish the objects mentioned above, the present inventionprovides a mesoporous platinum electrode for detecting biochemicalsubstrate, comprising an electrode and a mesoporous platinum layercovering the surface thereof.

The present invention provides a method for detecting a biochemicalsubstrate comprising (a) obtaining a mesoporous platinum electrodeincluding a electrode with a mesoporous platinum layer covering asurface thereof, (b) contacting a sample solution expected to containthe biochemical substrate with the mesoporous platinum electrode, (c)detecting a response current generated by applying a voltage to themesoporous platinum electrode.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the generated response current value when glucose, ascorbicacid, and acetamidophenol respectively is contacted with the mesoporousplatinum electrode; and

FIG. 2 is the calibration curve showing current generated relative toglucose concentration in the mesoporous platinum electrode.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is related to a method of detecting a biochemicalsubstrate using a non-enzymatic biosensor, and more particularly, to amethod of quantitating glucose concentration by monitoring the oxidationof glucose with a mesoporous platinum electrode. Examples of thebiochemical substrate include glucose, other saccharides (galactose,fructose, lactose, maltose, sucrose, etc.), electrochemically activeproteins (containing redox active group like hemin, Cu(II), FeS, flavinmononucleotide, flavin adenine dinucleotide, and disulfie), and aminoacids (like Tyr and Trp).

The biosensor of the present invention includes a mesoporous platinumelectrode that comprises an electrode and a mesoporous Pt layer coveringthe surface thereof. The biosensor can further contain a means thatprovides a potential to the electrode, or a means that can detect anamperometric response of the electrode.

The electrode can be a noble metal such as carbon, platinum, gold, orsilver, or a metal resistive to acid such as stainless steel.

The roughness of mesoporous Pt electrodes is smaller than the scale ofthe chronoamperometric diffusion field in most cases. Since thediffusion layers reach several micrometers from the electrode surface inmilliseconds, reactants inside the mesopores (of 2-50 nm in diameter)are depleted in diffusion-controlled electrochemical systems. As aresult, for rapidly oxidizable and/or reducible reactants, the faradaiccurrent is just proportional to the apparent geometric area of theelectrode within milliseconds regardless of the mesoporous roughnessafter a potential step. On the other hand, faradaic current associatedwith kinetic-controlled electrochemical events is sensitive to thenanoscopic area of the electrode rather than its geometric area. Thus,the mesoporous platinum electrode can be used to selectively enhance thefaradaic current from a sluggish reaction.

The mesoporous platinum layer can be fabricated by various methodsaccording to the known mesoporous material preparation method, andpreferably it can be fabricated by electrodeposition from a hexagonalliquid crystal phase comprising a non-ionic surfactant (octaethyleneglycol monohexadecyl ether, C₁₆EO₈). The electrodeposited mesoporous Ptlayer is formed uniformly on the metal electrode.

The mesoporous platinum layer has pores with diameters of 2-50 nm. It isdesirable that the roughness factor (the ratio of real surface area togeometric area) is as large as possible to enhance the selectivity andsensitivity for glucose. Practically the thickness of the layer is20-5000 nm. If the thickness is less than 20 nm, a mesoporous structureis not sufficiently formed and the selectivity for glucose can be poor.If the thickness is more than 5000 nm, pores can be blocked during thegrowth of the mesoporous Pt. The size of pores and thickness of the wallbetween pores is regulated by the length of hydrophobic and hydrophilicchains, so it is important to use adequate surfactant.

The method for detecting a biochemical substrate of the presentinvention comprises:

(a) obtaining a mesoporous platinum electrode including a electrode witha mesoporous platinum layer covering a surface thereof;

(b) contacting a sample solution expected to contain the biochemicalsubstrate with the mesoporous platinum electrode; and

(c) detecting a response current generated by applying a voltage to themesoporous platinum electrode.

The sample solution can be all kinds of body fluids from humans oranimals, and it can further contain a phosphate buffered solution (pH7.4) containing NaCl. The biochemical substrate can be measured in asample of water, blood, urine, serum or PBS buffer.

The adequate range of the applying voltage is preferably between −0.1and 0.5 V vs. a reference electrode. If the potential is outside thisrange, it may be difficult to measure the glucose concentration becauseother electrochemical processes such as adsorption/desorption ofprotons, formation of a platinum oxide layer, and oxygen reduction mayoccur significantly. The reference electrode prefers Ag/AgCl.

The current produced is proportional to biochemical substrate present inthe sample from a range of about 0 to 200 mM.

In one embodiment, the mesoporous platinum electrode of the presentinvention shows a linear response to glucose concentration (FIGS. 1 and2), and it shows a very low sensitivity to interfering materials. Thatis, the mesoporous platinum electrode can selectively respond to theglucose oxidation reaction.

The mesoporous surface of platinum offers a few attractive features thathave not been shown in any earlier studies. Firstly, it showsnon-enzymatic selectivity over representative interfering species.Although Sun et al.(Sun, Y.; Buck, H.; Mallouk, T. E. Anal. Chem. 2001,73, 1599-1604.) reported the Pt₂Pb alloy electrode insensitive tointerfering materials, it was achieved simply by lowering potentialwhere the interfering materials are not oxidized. Therefore, themesoporous platinum electrode of the present invention is more effectivebecause selectivity for glucose is evaluated at a potential that allowsthe oxidation of the interfering material. Considering physiologicallevel of glucose (3-8 mM) and interfering agents (0.1 mM), mesoporousplatinum electrode retains sufficient selectivity for clinicalapplication, even without enzyme or additional outer membrane.

Secondly, the function of the mesoporous surface is almost free fromdeterioration in the presence of chloride ion, even in the PBS level(0.15 M). Most of electrochemical sensors based on novel metals fornon-enzymatic glucose detection lose almost entire activity by poisoningby blocking materials, especially chloride ion. Concerning the highconcentration of chloride ion in physiological fluids, the excellentperformance of this simple system encourages us to find new breakthroughtoward enzyme-free chemical sensors.

Finally, mesoporous platinum electrode is mechanically and chemicallystable, and its surface can be easily regenerated by electrochemicalcleaning. It is expected that mesoporous platinum electrode can beembedded inside the microchannels on sophisticatedly engineered chips tocombine with microfluidics.

Examples of the present invention are demonstrated below. The examplesbelow are to illustrate the invention, and the present invention is notlimited by the examples.

EXAMPLE 1 Fabrication of Mesoporous Pt Electrode

C₁₆EO₈ (0.42 g), distilled water (0.29 g), and hydrogenhexachloroplatinate hydrate (0.29 g) were mixed, and the temperature wasraised to 80° C. until the mixture became transparent and homogeneous.Pt electrodes were inserted into the homogeneous mixture, and thetemperature was lowered to room temperature (˜23-26° C.). At this stagethe mixture became a highly viscous liquid crystalline phase. Platinumdeposition was carried out on a polished platinum rod electrode at aconstant potential (−0.06 V vs. Ag/AgCl). The resulting mesoporous Ptelectrode was placed in distilled water for 1 hr to extract C₁₆EO₈.After the extraction was repeated 3-4 times, electrochemical cleaningwas performed using a cycling potential between +1 and −0.45 V versusAg/AgCl in 0.5 N sulfuric acid until reproducibly identical cyclicvoltammograms were obtained.

The prepared mesoporous Pt electrode had a hexagonally arranged porestructure with a diameter of 2.5 nm and a wall thickness of 2.5 nm. Thecharacteristics of the mesoporous Pt were determined as follows.

-   -   Roughness factor of 72.5    -   Specific area, of 37 m²/g with roughness factor of 72, assuming        ˜30% of faradaic efficiency during electrodeposition process.    -   Mirror-like surface attributed to its excellent flatness        reported to be 20+5 nm over a mm² area of a 300 nm thick film    -   Single X-ray diffraction peak at 1.68 Å (2θ) corresponding to a        pore-pore distance of 6.1 nm    -   Diffusion characteristics same as those of a well-polished Pt        (Pt-s) surface

EXAMPLE 2 Quantitation of Glucose Concentration using Mesoporous PtElectrode

A current was measured using the mesoporous Pt electrode immersed in aphosphate buffered saline solution (0.1 M phosphate, 0.15 M NaCl, pH7.4, 37.2±0.2° C.) at 0.4 V vs Ag/AgCl as glucose, ascorbic acid, andacetamidophenol were added. The same measurement was done using polishedsmooth Pt as a control.

FIG. 1 shows current density vs time curves of responses of (a)mesoporous Pt electrode (roughness factor, 72) and (b) Pt-s (roughnessfactor, 2.6) to glucose, ascorbic acid (AA), and acetamidophenol (AP) at+0.4 V vs Ag/AgCl in aerated PBS (pH 7.4, 37.2±0.2° C.). Glucose,ascorbic acid, and acetamidophenol were added at the points indicated byarrows to the concentrations mentioned.

In amperometric responses of the mesoporous Pt electrode to glucose; AAand AP in phosphate buffered saline (PBS) solution containing 0.1 Mphosphated and 0.15 M NaCl (pH 7.4, 37.2±0.2° C.), the mesoporous Ptelectrode responds to glucose sensitively whereas Pt-s shows almost nosignal in the range of 1-20 mM. In addition, the mesoporous Ptselectively detected glucose rather than other interfering materialssuch as ascorbic acid and acetamidophenol. On the other hand,sensitivity for glucose was very low in comparison with the interferingspecies on smooth Pt.

FIG. 2 is a calibration curve showing the response relative to glucoseconcentration on the mesoporous Pt electrode. The signals weredetermined in quiescent solution 100 s after glucose was added to thesolution during stirring.

Table 1 shows the responses of mesoporous Pt and smooth Pt to glucoseand interfering materials.

TABLE 1 Electrode Glucose^(a) Ascorbic acid^(b) Acetamidophenol^(b)Mesoporous Pt 9.6 4.4 1.1 Smooth Pt 0.039 10 0.75 ^(a)Sensitivity (μAcm⁻² mM⁻¹) for glucose. ^(b)Signals for 0.1 mM (μA cm⁻²).

In FIG. 2 and Table 1, the mesoporous Pt electrode shows sensitivity of9.6 μAcm⁻²mM⁻¹, and the smooth Pt shows 0.039 μAcm⁻²mM⁻¹, in the glucoseconcentration range of 0-10 mM. Thus, the mesoporous Pt enhanced thesensitivity for glucose by 250 times that of the smooth Pt. The glucoseconcentration in a sample can be calculated using the calibration curvein FIG. 2.

As mentioned above, a mesoporous platinum electrode including anelectrode and a mesoporous platinum layer covering the surface thereofof the present invention can be applied to measure the glucoseconcentration quantitatively, and selective detection for glucose can berealized because the mesoporous platinum excludes interference byinterfering species. Furthermore, application of the mesoporous platinumas non-enzymatic glucose sensor largely enhances the selectivity,sensitivity, and stability in comparison with the previous glucosesensors.

1. A method for detecting glucose comprising: obtaining a mesoporousplatinum electrode in which the mesoporous platinum electrode comprisesan electrode and a mesoporous platinum layer covering the surfacethereof and in which the mesoporous platinum electrode is anon-enzymatic electrode without any enzyme immobilized thereon thatreacts with the glucose to produce an electrical signal; contacting asample solution expected to contain the biochemical substrate with themesoporous platinum electrode; detecting a response current generated byapplying a voltage to the mesoporous platinum electrode.
 2. The methodof claim 1, wherein the electrode is a noble metal or an acid-resistivemetal.
 3. The method of claim 1, wherein the electrode is selected fromthe group consisting of carbon, platinum, gold, silver, and stainlesssteel.
 4. The method of claim 1, wherein the mesoporous platinum layerhas a thickness of 20-5000 nm.
 5. The method of claim 1, wherein thecurrent is measured amperometrically.
 6. The method of claim 1, whereina range of the applying voltage is between −0.1 and 0.5 V vs. areference electrode.
 7. The method of claim 6, wherein the referenceelectrode is Ag/AgCl.
 8. The method of claim 1, wherein the currentgenerated is proportional to the glucose present in the sample from arange of 0 to 20 mM glucose.